Blood glucose levels (blood sugar levels) are an important marker of diabetes. An SMBG (self-monitoring of blood glucose) device using an electrochemical biosensor is widely used for managing blood glucose levels in patients with diabetes. Enzymes that catalyze glucose as a substrate, such as glucose oxidase (GOD), have conventionally been used for biosensors employed in SMBG devices. However, GOD is characterized by the use of oxygen as an electron acceptor. Thus, SMBG devices using GOD may influence the measurement of dissolved oxygen in a sample, precluding accurate measurement.
Other enzymes that use glucose as a substrate, but do not use oxygen as an electron acceptor include various glucose dehydrogenases (GDHs). Specifically, GDH (NAD (P)-GDH) that uses nicotinamide dinucleotide (NAD) or nicotinamide dinucleotide phosphate (NADP) as a coenzyme and GDH(PQQ-GDH) that uses pyrroloquinoline quinone (PQQ) as a coenzyme were found, and have been used for the biosensors of SMBG devices. However, NAD (P)-GDH has a problem that the enzyme is unstable and requires the addition of coenzyme. PQQ-GDH has a problem that sugar compounds other than glucose in a sample affect measurements, precluding accurate measurements, because it also reacts with sugar compounds other than glucose to be measured, such as maltose, D-galactose, and D-xylose, because of low substrate specificity.
According to a recent report, during the measurement of the blood glucose level of a patient with diabetes, who received infusion with an SMBG device using PQQ-GDH as a biosensor, PQQ-GDH also reacted with maltose contained in an infusion, raising a measured value as compared with the actual blood glucose level, and the patient developed hypoglycemia due to treatment based on this value. In addition, similar events may occur in patients who participate in a trial on galactose tolerance or xylose absorption (see, for example, Non-patent document 1). In response to this, the Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare conducted a cross-reactivity test to investigate the effects of the addition of each sugar into a glucose solution on blood glucose measurements. When maltose was added at 600 mg/dL, D-galactose at 300 mg/dL, and D-xylose at 200 mg/dL, measurements with a blood glucose measurement kit using the PQQ-GDH method were 2.5-3 times higher than the actual glucose level. Specifically, maltose, D-galactose, and D-xylose that may exist in a measurement sample preclude accurate measurement. The development of GDH that allows specific glucose measurement with high substrate specificity without being affected by sugar compounds that cause measurement errors is desired.
Under the above circumstances, GDHs using coenzymes other than those described above have attracted attention. For example, although the substrate specificity has not been described in detail, reports were published regarding a GDH derived from Aspergillus oryzae (see, for example, Non-patent documents 2-5). In addition, a glucose dehydrogenase using flavin adenine dinucleotide (FAD) from Aspergillus as a coenzyme (FAD-GDH) has been disclosed (see, for example, Patent documents 1-3). An FAD-GDH derived from Aspergillus, with reduced effects on D-xylose, has also been disclosed (see, for example, Patent document 4).
As described above, some FAD-GDHs having low reactivity with one or several sugar compounds other than D-glucose are known. However, no flavin-bound GDH having sufficiently low reactivity with all of maltose, D-galactose, and D-xylose is known. In addition, no flavin-bound GDH that allows accurate measurement of glucose levels in the presence of D-glucose, maltose, D-galactose, and D-xylose without being influenced by sugar compounds thereof is known. In addition, neither method nor means for efficiently producing a flavin-bound GDH having such excellent substrate specificity has been reported.
Methods for preparing a transformant by introducing an enzyme gene of interest into a suitable host and producing the enzyme by culturing the transformant is conventionally known as a means for efficiently producing a useful enzyme. Particularly, a method for introducing a gene into E. coli is widely used as a means for efficiently producing a substance. However, there are few findings regarding recombinant FAD-GDH production in E. coli. Only a method for recombinant expression by introducing an FAD-GDH gene derived from Aspergillus or Penicillium into E. coli K12 strain host is disclosed (for example, see, Patent document 5).
In expressing a gene by introducing it into a host, the efficiency actually varies with genes and heterologous hosts. Of note, in introducing a gene into a heterologous host, particularly, introducing a eukaryotic gene into E. coli, there may often be problems with introduction or expression in the host even using a known method with reference to the findings of the same kind of enzyme. E. coli has no a post-translational modification system. Thus, in general, expressing the activity of an enzyme derived from a eukaryotic organism (e.g., fungi) in E. coli is often difficult when the enzyme activity requires post-translational modification. For example, when an enzyme derived from fungi is expressed in E. coli, it causes an insoluble inclusion body in most cases.
Under such circumstances, it is industrially useful to find a combination of gene, host, and introduction method, which facilitates the expression and efficient production of an enzyme, derived from a eukaryotic organism (e.g., fungi), in E. coli. Besides an FAD-GDH having excellent properties, a technique for efficient production of an enzyme having such excellent properties in E. coli, advantageous for industrial enzyme production, is strongly demanded.